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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

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2006 - 20092
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Author: Naveen Kumar × End date: 2009 × Start date: 2004 ×
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    Detection of anti Non-Structural protein 3A (NSP-3A) antibodies in sera of cattle and buffaloes vaccinated with FMD vaccine in Haryana
    (LUVAS, 2009) Naveen Kumar; Batra, S.K.
    Foot-and-mouth disease (FMD) is a highly infectious and contagious vesicular disease affecting cloven-hoofed animals. FMD virus (FMDV) belongs to the genus Aphthovirus in the Picornaviridae family and includes seven serotypes, O, A, Asia1, C, and SAT1, -2, and -3. The circulation of FMDV in an animal population imposes severe restrictions on the movement of animal products and consequently on international trade. FMD is endemic in many parts of Asia, Africa, and South America. In India, mass vaccination with trivalent vaccine (O, A, and Asia1) has been adopted for control and eradication for FMD. Due to endemicity of the disease, FMD control programme has been launched in selected states including Haryana to create FMD free zones. The major constrain to attain the FMD free status is the carrier animals. A carrier of FMDV is defined as an animal from which live-virus can be recovered from scrapings of the oropharynx after 28 days following infection (Sutmoller and Gaggero, 1965). FMD vaccines do not provide sterile immunity and animals can become clinically or sub clinically infected and ultimately become carriers of the virus, which is considered a threat for spread of the disease to other susceptible animals (Cox et al., 2005, 2006; Doel et al., 1994; Hargreaves et al., 2004; Kitching, 2002; Moonen and Schrijver, 2000). Therefore, to regain FMD-free status and re-enable international trade, post vaccination surveillance is required to demonstrate the absence of persistent infection in a vaccinated population (Anon. 2004). In the present study, a total of 2000 serum samples were used in Liquid phase blocking ELISA (LPBE) for vaccinal antibody response and 3A-NSP ELISA for carrier/persistent infection of the FMDV. On analyzing those serum samples for vaccinal immune response using LPBE, it was observed that there was no significant difference in FMD-CP (FMD-control programme) districts and ASCAD (Assistance to States for Control of Animal Diseases programme) districts of Haryana irrespective of biannual and annual vaccinations respectively. Also same serum samples were used for detection of antibodies to 3A-NSP where it was 29 observed that there was no significant difference in prevalence of anti-3A NSP antibodies in serum samples of cattle and buffaloes taken together from FMD-CP and ASCAD districts of Haryana. It was found that anti-3A NSP antibodies positive cattle were significantly higher than buffaloes (p<0.001) in both the FMD-CP and ASCAD districts. Further isotyping of 3A-NSP specific antibodies to know which isotype of immunoglobulin IgG predominated during the FMDV infection revealed that both isotypes i.e. IgG1 and IgG2 were prevalent in both the FMD-CP and ASCAD districts.
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    Molecular and immunological studies on persistence of FMD virus
    (LUVAS, 2006) Naveen Kumar; Sharma, R.
    The present study was undertaken to develop and standardise a test for differentiation of Foot-and-Mouth Disease (FMD) vaccinated and infected animals (DIVA strategy). This test, which was developed at Freidrich Loeffler Institute, Greifswald, Insel Riems, Germany, was subsequently used for DIVA strategy in Haryana (India). Apart from this, carrier state was also studied in vaccinated-challenged and naïve-challenged cattle after an experimental infection. The Non-structural-protein (NSP) 3A of FMDV was cloned and expressed in baculovirus expression system. The identity of the expressed protein was analysed in indirect immunofluorescence and Western blotting. The RGS 6X His fusion protein, expressed by recombinant baculovirus was purified by Ni-agarose affinity chromatography and further tested for its identity by SDS-PAGE. The purified protein was used to immunise mice for development of monoclonal antibodies against FMDV NSP 3A. Hybridomas were produced by fusion of spleen cells of immunised mice and myeloma cell line. The culture supernatants from hybridomas were tested for their reactivity and specificity to NSP 3A by ELISA and immunofluorescence respectively. Two good IgG1 secreting clones (5G1 and 8H8) which were giving a titer of 1:150 (pooled) were selected for 3ANSP-ELISA. The indirect monoclonal antibody based 3ANSP-ELISA was standardised by using baculovirus expressed NSP 3A and monoclonal antibodies developed in mice. By using this indirect ELISA, 439 naive cattle sera, 272 FMDV once vaccinated cattle sera, 382 reference positive cattle sera and 33 FMDV multiple vaccinated sera were tested. At 20% cut off and 98.18 % specificity, the test sensitivity reached to 99.22 %. One Homologous challenge experiment (HoCE) (A Iran96 vaccine /A Iran96 challenge virus) and two Heterologous challenge experiments (HtCEs) (A Iraq22 vaccine/A Iran96 challenge virus and it’s vice versa i.e. A Iran96 vaccine/ A Iraq22 challenge virus) were performed. In HoCE, vaccinated-challenged animals were completely protected (PD50>32). In 1st HtCE, the vaccine offered good protection (PD50>6) but vice versa of challenge and vaccine virus in another HtCE (A96 vaccine and A22 challenge virus) could not offer protection (PD50<2). High potency vaccine induced high antibody titers (>3 in most cases) in all the three experiments. The carrier state was studied at successive stages after infection (15, 19, 24 and at least up to 28 days post-infection; dpi) by virus isolation (plaque test) & nucleic acid detection (Real-Time PCR) from oropharyngeal fluid, detection of secretory IgA in saliva (IgA-ELISA) and antibody response against NSPs in serum (NSP-ELISA). The NSP based detection of carriers was found most sensitive, followed by detection of viral RNA, secretory IgA response and virus isolation. High potency vaccine was found to reduce the carrier state in HoCE but in HtCEs, inspite of giving good protection in certain cases, it could not prevent the development of carrier state. A significant reduction (P<0.05) in the carrier cattle and buffalo (anti-NSP antibody positive) was observed 2 years after launching FMD control programme in Haryana. Population of carrier cattle was found significantly (P<0.01) higher than buffaloes, both before (P<0.01) and after (P<0.01) launching FMD control programme in the state.